Glycoconjugates are the major components of the external glycocalyx of cells and, to a large extent, determine their surface properties. Lipid glycoconjugates predominate in the nervous system, and of these the gangliosides have been the major focus of interest (aside from cerebroside of myelin). This application is designed to fill a major gap in this area by beginning a systematic study of neutral glycosphingolipids (GSLs), particularly in the developing nervous system where recent studies have shown these substances to be considerably more prominent than had been previously recognized. Furthermore, they undergo marked changes as development progresses. We shall begin with a study of whole brain of 2 species --rat and chicken -- and characterize the types and quantities of neutral GSLs at various stages of embryonic and perinatal growth. This should provide an abundance of neutral GSLs for structural characterization, thereby laying the groundwork for more detailed studies of individual cell types in culture. Primary cultures of neurons, astrocytes and oligodendroglia will be obtained from rat and chick brain and neutral GSLs labeled to determine molecular patterns of each cell type at various stages of development. Growth cone membranes, isolated from embryonic rat brain by a recently developed procedure, were found to have appreciable neutral GSLs, and further characterization of these substances will be a major objective. We intend to follow up a recent discovery of our group that sensory neurons of rat sciatic nerve contain a considerable amount of neutral GSLs (in addition to gangiosides) - - the only such example to date for mature neurons; use of radiolabeling techniques and the fact that these substances undergo axonal transport will assist us in identifying these substances. The biological role of neutral GSLs will be examined in various ways: (a) observation of developmental changes in vivo and in vitro, (b) insertion of these lipids into membranes of neural cells in vitro, to determine their effect on development, and (c) the use of interventive agents (e.g. antibodies) to hinder development at specific stages. Insertion into membranes will be aided by glycolipid transfer proteins, now being isolated and characterized in our laboratory. Further study of such proteins will be part of this project.